Approximately 20-30 million Americans are affected by Mendelian genetic disorders with broad clinical consequences including congenital heart disease, congenital bone diseases, inherited skin diseases, hereditary neurological disorders, hereditary cancers, and others. Over the last several years, high-throughput, whole-exome sequencing has been used for molecular diagnosis and as a research tool for discovery of new disease-causative gene(s). Since the first successful application of this technology six years ago, the fundamental genetic bases for over 100 Mendelian diseases have been identified. Despite these advances, the overall success rate for human Mendelian disease gene discovery by whole-exome sequencing remains at slightly less than 50 percent. In contrast to these clinical cases, the discovery of Mendelian disease genes in mice is powered by genetically defined inbred strain backgrounds, large consanguineous pedigrees for segregation analysis, and disease modeling through the use of exciting new CRISPR/Cas9 approaches and more traditional genetic engineering techniques. With these allied technologies, the application of whole-exome sequencing in recent years has increased the rate of mutation discovery in mouse by nearly ten fold. Still, as in humans, the success rate for Mendelian disease gene discovery in the mouse is only slightly better than 50 percent. Possible limitations of whole-exome sequencing for disease gene discovery in both the mouse and human genomes include shortcomings of variant calling tools, insufficient resources describing `normal' genome variation, and the likely existence of regulatory mutations that, residing outside of protein-codin regions, escape detection by exome-sequencing. With the promise of exploring and surmounting these limitations, our long-term goal is to develop genomic resources that facilitate functionalization of both the coding and non-coding portions of the mouse genome through forward genetic discovery approaches and reverse genetic validation. More specifically, the objectives of this proposal are to expand the scope of gene discovery beyond exome-imposed limitations, and to use these data to develop a mouse genome variation database, and other optimized resources, that will deliver improved mutation discovery success rates. Moreover, we will apply these new technologies to explore de novo mutations and perinatal lethal phenotypes in a large population of mouse neonates, as well as identifying the heritable molecular lesions in established, genetically defined Mendelian disease models. In both settings, we will validate and model the mutations using modern CRISPR/Cas9 and other genetic engineering approaches.